Prolactin is a pituitary hormone involved in numerous reproductive and non-reproductive processes. Prolactinomas are the most common of the hormone-secreting pituitary tumors and hypersecretion of prolactin (pRL) is the major neuroendocrine-related pathology associated with female infertility. Unlike most endocrine cells, lactotropes require continuous inhibition to keep their secretory activity under control. The majority of this inhibition comes from hypothalamic dopamine (DA). This inhibitory influence coordinates with the stimulatory actions of numerous secretagogues to elicit surges of PRL under certain physiological conditions. Both, in turn, are modulated by the ovarian steroid, estrogen. The combined or sequential effects of these regulators on PRL release are poorly understood. Moreover, subpopulations of lactotropes may exhibit differences in responsiveness to the various modulators. Thus, definitive conclusions regarding the physiological roles of putative prolactin regulators remain elusive. Elucidation of the cellular events mediating the actions of PRL regulators is required for understanding their physiological role in PRL secretion. In addition, identification of subpopulations of lactotropes differentially affected by these regulators is inherent to this understanding. Relatively little is known about the ionic mechanisms involved in excitation-secretion coupling in endocrine cells beyond a requirement for Ca2+ and the presence of regenerative electrical responses in the cell membrane. Investigation of the functional coupling between l) membrane electrical events, 2) cytosolic Ca2+ and 3) the exocytotic release of PRL is the first step to understanding the physiology of PRL secretion. This revised proposal outlines a multidisciplinary experimental approach to the study of mechanisms in the regulation of PRL release from lactotropes dissociated from normal rat anterior pituitary tissue. Three state-of-the-art technological advances make this investigative approach possible in normal endocrine cells: l) positive identification of viable, PRL-secreting cells in mixed cell populations using the reverse hemolytic plaque assay; 2) recording of whole cell and single channel conductances using giga-seal patch clamp techniques; and 3) direct measurement of cytoplasmic Ca2+ concentration in single cells using fluorescent microscopy and dynamic imaging of the calcium indicator fura-2. Using these techniques on cells in vitro, the short-term objectives of this project are to identify and characterize the ionic and biochemical messages transducing the actions of DA and estrogen on PRL release. Characterization of the mechanisms underlying the action of individual modulators will establish the necessary basis for understanding integrated neuroendocrine function during physiological changes in prolactin secretion. In addition, this research will provide a framework in which to examine cellular and membrane properties and abnormalities associated with pathologies of hormone secretion. As such, these studies have a direct bearing on the regulation of fertility and reproduction and in the clinical management of hyperprolactinemia.